PHYSIOLOGY OF ALKALIPHILIC
SULFUR-OXIDIZING BACTERIA FROM
PHYSIOLOGY OF ALKALIPHILIC
SULFUR-OXIDIZING BACTERIA FROM
SODA LAKES
PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de Technische Universiteit Delft,
op gezag van de Rector Magnificus Prof. dr. ir. J.T. Fokkema, voorzitter van het College voor Promoties,
in het openbaar te verdedigen op maandag 1 november 2004 om 10.30 uur door
Horia Leonard BANCIU
Master degree in Cell and Molecular Biology, “Babeş-Bolyai” University, Cluj-Napoca, România
Dit proefschrift is goedgekeurd door de promotor: Prof. dr. J. G. Kuenen
Samenstelling promotiecommissie:
Rector Magnificus voorzitter
Prof. dr. J. G. Kuenen Technische Universiteit Delft, promotor Prof. dr. A. Oren Hebrew University, Jerusalem, Israel Prof. dr. C. N. Tarba Universitatea Babeş-Bolyai
Cluj-Napoca, Romania Prof. dr. J. T. Pronk Technische Universiteit Delft Prof. dr. ir. M. C. M. van Loosdrecht Technische Universiteit Delft
Dr. G. Muyzer Technische Universiteit Delft
Dr. D. Y. Sorokin Insitute of Microbiology, RAS, Russia Prof. dr. J. P. van Dijken Technische Universiteit Delft, reservelid
Dr. Dimitry Y. Sorokin has provided substantial guidance and support in preparation of this thesis.
This study was carried out in the section of Environmental Microbiology at the Department of Biotechnology, Kluyver Laboratory for Biotechnology, Faculty of Applied Sciences, Delft University of Technology, The Netherlands.
Physiology of alkaliphilic sulfur-oxidizing bacteria from soda lakes/ Horia Leonard Banciu: Delft University of Technology, Faculty of Applied Sciences.
Thesis Delft University of Technology.-With ref.-With summary in Dutch
This work was financially supported by the Dutch Technology
Foundation (STW) by the contract DST 60.4653.
ISBN 90-77595-87-2
Chapter 1 ... 1
General introduction Chapter 2 ... 31
Growth physiology and competitive interaction of obligately chemolithoautotrophic, haloalkaliphilic, sulfur-oxidizing bacteria from soda lakes Chapter 3 ... 53
Sodium salts requirement for the growth and activity in Thioalkalivibrio versutus strains from soda lakes: halophiles vs. natronophiles Chapter 4 ... 67
Growth kinetics of the haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio versutus strain ALJ 15 in continuous culture Chapter 5 ... 89
Thioalkalivibrio halophilus sp. nov., a novel obligately chemolithoautotrophic facultatively alkaliphilic and extremely salt-tolerant sulfur-oxidizing bacterium from a hypersaline alkaline lake Chapter 6 ... 111
Membrane properties and compatible solutes composition of obligately chemolithoautotrophic alkaliphilic sulfur-oxidizing bacteria from soda lakes Chapter 7 ... 133 General discussion Summary/ Samenvatting ... 143 Curriculum Vitae ... 149 List of publications ... 151 Acknowledgements ... 153
General introduction
1. Saline environments
Extreme environments are widely distributed on Earth and they comprise the immense desert regions, the surface and deep-sea active volcanic areas, the thermal and often acidic springs and lakes, the Arctic and Antarctic ice shields, the permafrost and glaciers, the saline and/or alkaline soils and lakes etc. Despite the apparently adverse physico-chemical conditions, these extreme environments are densely populated by microorganisms. In the past decades a growing number of new genera and species of extremophilic microorganisms have been discovered and described. However, in spite of these recent advances, the cultivated bacteria only make up a small percentage of the microorganisms known to be present in these environments.
The present thesis concerns one particular segment of these extreme milieus, namely saline and alkaline environments. Most saline and/or alkaline lakes are located in the arid areas in Middle East and Central Asia, Eastern Africa, the western and northern part of the U.S.A and Central Australia. Remote saline lakes have been also found in other part of the world, such as the hypersaline Antarctic lakes (Lawson et al., 2000). The saline lakes may have originated from a complex interaction of biogeochemical, geographical and climatic conditions. Based on these particularities acidic, neutral or alkaline saline lakes can occur. Saline lakes may contain water permanently, intermittently or transiently. They range from deep to shallow, from small to extremely large (Great Salt Lake, Dead Sea).
Based on their marine or continental origin, the saline lakes are divided into thalassohaline (with the same salt composition as seawater) and athalassohaline lakes (with a salt composition different from that of the seawater) (Table 1) (Oren, 2002).
Table 1. Concentration of ions in thalassohaline and athalassohaline brines (modified after Grant et al., 1998; Zavarzin et al., 1999; Imhoff et al., 1979)
Concentration (g/l) Ion Seawater Great Salt Lake, U.S.A. Dead Sea Big Soda Lake, U.S.A. Lake Hadyn, Siberia Lake Magadi, Kenya Lake Zugm-Wadi Natrun, Egypt Na+ 10.8 105.0 39.7 8.1 3.9 161.0 142.0 Mg2+ 1.3 11.1 42.4 0.32 0.28 0 0 Ca2+ 0.4 0.3 17.2 0.15 0 0 0 K+ 0.4 6.7 7.6 0.005 0 2.3 2.3 Cl- 19.4 181.0 219.0 7.1 2.6 111.8 154.6 SO42- 2.7 27.0 0.4 5.8 3.8 16.8 22.6 CO3 2-/HCO3- 0.34 0.72 0.2 4.1 2.4 23.4 67.2 pH 8.2 7.7 6.3 9.7 8.4 11.0 11.0
The neutral saline lakes (pH 6-8) contain NaCl as the major salt and their buffering capacity is low. On the other hand, the alkaline saline (soda) lakes (pH 9-11) are characterized by the presence of large amounts of sodium carbonates (Na2CO3 + NaHCO3) that confer the water a high buffering capacity (Fig. 1). Naturally occurring alkalinity is usually associated with salinity (Grant and Tindall, 1986). Other major ions found in salt lakes are Mg2+, Ca2+ and K+ as cations, and SO
42- and Br- as anions. In the soda lakes, one of the major chemical characteristics is the lack of solubilized divalent cations (Mg2+, Ca2+) due to their strong tendency of precipitation as carbonates under alkaline conditions. The removal of divalent cation carbonates drives the solubilization of sodium or potassium carbonates, thus increasing the monovalent cation concentration. In this way brines are formed. In some parts of the world, the shallow lakes may end as a layer of solid rock (trona – crystalline sodium sesquihydrate, Na2CO3•NaHCO3•2H2O) by evaporation during dry seasons. A broad range of intermediate saline and/or alkaline lakes occur by the mixing of the minerals in various ratios. Several examples of saline lakes an their chemical composition are presented in Table 1.
In general, at a salt concentration higher than that found in seawater (35 g/l, w/v), the lake is considered highly saline or hypersaline (Grant et al., 1998). The saline lakes have attracted the attention of investigators due to their unique chemical and biological features. Soda lakes are considered as models of ancient Martian or Archaean terrestrial aquatic
biotopes (Kempe and Degens, 1985; Kempe and Kazmierczak, 1997). The (hyper)saline lakes are populated mostly with halophilic neutrophilic organisms while the alkaline saline lakes are the habitats of haloalkaliphilic species. The organisms living in such environments possess special adaptation mechanisms that make them interesting for industrial application (Margesin and Schinner, 2001).
Salt lake type 1 pH 7-8 e.g. Great Salt Lake,
Utah pH 6-8 CaSO4 ↓, CaCO3 ↓, MgCO3 ↓, CaMg(CO3)2 ↓ Na+ K+ Ca2+ Mg2+ HCO3- CO3 2-SiO2 SO42- Cl -Low Ca2+ and Mg2+ High Ca2+ and Mg2+ High Ca2+ Na+ Cl -CO3 2-Na+ Mg2+ Cl -Salt lake type 2
pH 6-7 e.g. Dead Sea Na+
Cl
-Soda lake pH 10-11 e.g. Lake Magadi, Kenya
Figure 1. Schematic representation of the genesis of acidic, neutral and alkaline saline lakes (modified after Grant et al., 1998; Jones et al., 1994)
2. Diversity of halophilic microorganisms
In the past decades the studies revealed a large diversity of organisms that thrive in highly saline and alkaline lakes (Duckworth et al., 1996; Humayoun et al., 2003; Jones et al., 1998; Oren, 1994, 2002). Based on their salt tolerance and optimal salt concentration, the halophilic organisms can be grouped into several categories: halotolerants, low-salt
halophiles, moderate halophiles, borderline extreme halophiles, extreme halophiles and haloversatiles (Table 2) (Grant et al., 1998; Ventosa, 1989). The halophiles that have their pH optimum at alkaline values are called haloalkaliphiles. On the reverse, many alkaliphiles are low salt or nonhalophilic alkaliphiles (Hamamoto and Horikoshi, 1992). The term “natronophilic” (or “trona–loving“ organisms) has been suggested for the organisms living at high soda (carbonate/bicarbonate) concentrations rather than at high NaCl concentrations. This quality may apply to those haloalkaliphilic organisms isolated from soda lakes and growing optimally at high pH and high soda concentrations. In the following text we will use the terms “halophilic” or “halophiles” for those organisms that are at least low-salt halophiles (require a minimum of 0.2 M or 15 g/l salts).
Table 2. Categories of halophilic microorganisms (modified after Grant et al., 1998; Ventosa, 1989)
Salt concentration (M)
Category Range Optimum Example
Nonhalophiles 0-1.0 <0.2 Escherichia coli
Low-salt halophiles 0.2-2.0 0.2-0.5 Thioalkalimicrobium aerophilum Moderate halophiles 0.4-3.5 0.5-2.0 Desulfovibrio halophilus Borderline extreme halophiles 1.4-4.0 2.0-3.0 Halorhodospira abdelmalekii Extreme halophiles
2.0-5.2 >3.0 halophilic Archaea (e.g. Halococcus sp.) (Extreme) Halotolerants 0->1.0
(5.0) <0.2 Dunaliella
Haloversatiles 0->3.0 0.2-0.5 Halothiobacillus kellyi
Halophilic organisms are found in all three domains of life: Archaea, Bacteria and
Eucarya. A large nutritional and ecological spectrum of halophiles assures the natural
cycling of the elements in the saline environments. The taxonomy, physiology and molecular aspects of halophiles and halophilic adaptations have been extensively reviewed in the past (Grant et al., 1998; Kushner, 1989; Oren, 1986; 1999; 2002; Reed, 1986; Ventosa, 1989; Ventosa et al., 1998; Baumgarte, 2003).
Most of the halophilic organisms are prokaryotes and only a few are eukaryotes. The unicellular eukaryotes, such as green algae of genus Dunaliella can live up to saturation values of NaCl. The green algae are the trophic base for higher eukaryotes in the (hyper)saline lakes like the brine shrimps (Artemia franciscana, A. monica) and the brine flies (Ephydra gracilis, E. hians). Species of protozoa, fungi, invertebrates and plants tolerating high concentrations of NaCl have also been described (DasSarma and Arora, 2002).
The primary biomass producers in the soda lakes are mostly the haloalkalpihilic cyanobacteria that can produce up to 10 g C/ m2 day (Grant et al., 1990). The low saline soda lakes turn often into a red color because of seasonal blooming of halophilic cyanobacteria while the primary biomass producers in hypersaline soda lakes are both haloalkaliphilic cyanobacteria and anoxygenic phototrophic bacteria. The prolific development of haloalkaliphilic cyanobacteria (Cyanospira, Arthrospira, Spirulina,
Synechococcus) provide the trophic base for large populations of aquatic birds (Krienitz et
al., 2003; Zavarzin et al., 1999). Cyanobacteria are essential both for N2 fixation and for O2 production in the saline lakes.
Haloalkaliphilic phototrophic anoxygenic bacteria are represented mainly by purple sulfur bacteria of the genera Ectothiorhodospira and Halorhodospira. They use inorganic sulfur compounds like sulfide and elemental sulfur as electron donor for C fixation and for growth. The organic compounds are further mineralized through biological processes catalyzed aerobically by halophilic heterotrophic bacteria of the genus Halomonas (Duckworth et al., 2000). The final step in the organic matter degradation occurs primarily under anaerobic conditions. In principle all major metabolic group are represented among the halophiles. Aerobic phototrophic (e.g. Roseinatronobacter thioxidans) (Sorokin et al., 2000), phototrophic nonsulfur bacteria (e.g. Rhodobaca bogoriensis) (Milford et al., 2000), photoheterotrophic heliobacteria (e.g. Heliorestis daurensis, H. baculata) (Bryantseva et al., 1999, 2000) have been found in low-saline East African or Siberian lakes. At extreme salt concentrations (>150 g/l) several physiological groups of organisms could not be found: autotrophic methanogens, acetoclastic methanogens, dissimilatory sulfate-reducers that perform the complete oxidation of their substrate and autotrophic ammonia and nitrite oxidizers. According to Oren (1999) the reason for the absence of these nutritional groups at hypersaline conditions might be the bioenergetic constraints. It is speculated that since these organisms perform a less energetically efficient metabolism, they could not sustain an energetically expensive osmoregulation.
3. Sulfur cycle and trophic relationships among
haloalkaliphilic sulfur bacteria
Sulfur is one of the most important elements for sustaining life on Earth. The sulfur chemistry is complicated by the many oxidation states sulfur can assume (Table 3).
Table 3. Oxidation states of sulfur in common compounds (after Steudel, 2000; Brüser et al., 2000)
Oxidation state Compounds
-2 Dihydrogen sulfide H2S, hydrogen sulfide ion HS
-, sulfide ion S2- as in FeS; thiocyanate SCN --1 Disulfane H2S2; disulfide S2 2- as in pyrite FeS 2; thiosulfate sulfane S1-; polysulfides -S(S) nS
-0 Elemental sulfur Sn; organic polysulfanes R-Sn-R; polythionates -O
3S(S)nSO3
-+1 Dichlorodisulfane Cl-S-S-Cl +2 Sulfur dichloride SCl2; sulfoxylate SO2 2-+3 Dithionite S2O4
2-+4 Sulfur dioxide SO2; sulfite SO32-; bisulfite HSO3
-+5 Dithionate S2O62-; sulfonate RSO3-; thiosulfate sulfone SO3- +6 Sulfur trioxide SO3; sulfate SO42-; peroxosulfate SO5
2-Geochemically, sulfur is very abundant and several sources of production, emission or storage can be identified (Table 4). The biochemical significance of sulfur is tremendous. The origin of life has been linked with iron sulfide (pyrite) that becomes catalytically active at elevated temperature and at high pressures (Wächtershäuser, 1988; Cody et al., 2000; Martin and Russell, 2003). Sulfur plays a catalytical role in the iron-sulfur clusters within respiratory enzymes. Sulfur containing aminoacids (cysteine, cystine and methionine), sulfolipids, and many co-enzymes (glutathione, coenzyme A, biotin, lipoic acid) are essential for cell metabolism.
Table 4. Main sources of sulfur
Source of sulfur Dominant sulfur compound
Volcanic activity SO2
Biogenic emissions
(from vegetation, wetlands, lands)
H2S, dimethyl sulfide, carbonyl sulfide Biogenic emissions
from oceanic environments
SO42-, dim ethylsulfide Anthropogenic activities SO2 , SO3
Sulfur storage products Gypsum (CaSO4 • 2H2O),
metal sulfides, elemental sulfur (S0)
In the natural environment the element sulfur is part of a closed cycle with alternating oxidized and reduced sulfur species, in the organic and inorganic forms. The chemical sulfur cycle strongly interacts with biological activity resulting in utilization, transformation and storage of sulfur compounds (Figure 2). Nevertheless, the biological importance of sulfur compounds resides in their capacity to serve as electron donor and acceptor for anaerobic respiration or aerobic light-dependent CO2 reduction and moreover, as an aerobic energy source for ATP production (Lens and Kuenen, 2001).
The high salinity and hence high density of the hypersaline and many soda lakes explain why they are often hydrologically stratified. This often results in a layer of less saline water permanently covering the concentrated salt layer (known as meromixis). (Hollibaugh et al., 2001). Only the upper layers of the water contain oxygen and can support eukaryotic and aerophilic prokaryotic life. Due to the lack of mixing, the bottom waters are anoxic and at alkaline pH values, toxic inorganic compounds as sulfide or ammonia accumulate. The stratification of physical properties (e.g. temperature, light) or chemical parameters (dissolved oxygen, pH, salinity) is reflected in a stratification of microbial community.
Microorganisms involved in the sulfur cycle from saline and alkaline environments like soda lakes have been well studied. The haloalkaliphilic sulfur-oxidizing phototrophic and anoxygenic bacteria are members of the genus Ectothiorhodospira (E. haloalkaliphila,
E. vacuolata) (Imhoff et al., 1979; Tindall, 1980) and of the genus Halorhodospira sp. (H. halophila, H. halochloris, H. abdelmalekii) (Imhoff and Süling, 1996; Oren 2002).
Dissimilatory sulfur reduction Anaerobic oxidation by phototrophic bacteria Anaerobic oxidation by phototrophic bacteria Biological oxidation with O2 or NO3 -Chemical oxidation Biological oxidation with O2 or NO3 -Biological oxidation with O2 or NO3
-Dissimilatory sulfate reduction Mineralization processes Assimilatory sulfate reduction Sulfur deposits Sulfidic minerals (e.g. pyrite) Sulfate reserves (seawater) S0 S 2-SO4 2-Organic sulfur compounds
Figure 2. The biological sulfur cycle (Robertson and Kuenen, 1992). The thickened arrows indicate the oxidation processes that occur in the alkaliphilic SOB from soda lakes.
The halophilic sulfate-reducing bacteria are found within Desulfonatronovibrio sp. (D.
hydrogenovorans) (Zhilina et al., 1997) and Desulfonatronum (D. lacustre, D. thiodismutans) (Pikuta et al., 1998, 2003). Together with recently discovered obligately
chemolithoautotrophic sulfur-oxidizing bacteria of the genera Thioalkalimicrobium and
Thioalkalivibrio (Sorokin et al., 2001b, c, 2002), the heterotrophic sulfur-oxidizers and
sulfate-reducers constitute an ecologically balanced microbial community that ensures the recycling of sulfur in the saline and alkaline lakes (Fig. 3).
Depth Sediment/w ater in ter face Sedi ment O2 pres ent (e xces s) O2 lim iti ng D is so lv ed s ul fide present (e xc ess)
[H
2S]
O xy genic photot rophi c ba ct eria (c ya nobac teria ) Aerobi c che mohe terotrophic bact eri a Chem ol it hoaut otrophic sulf ur ba ct eri a Anox yg en ic phot ot rophi c (purpl e) sul fur ba cte ri a A nae robic su lf at e-re du cing bacteria SO 4 2-H2 S D iss ol ve d s ul fid e l imi ting Anaerobic D iss ol ve d s ul fid e l imi ting Anaerobic S 0, S O4 2-O rg anic sul fur input het erot rophi c ba cte ri a[O
2]
G as c oncent ra ti on (% of s at urat ion) 100 H2 SS 0, S O4 2-Depth Sediment/w ater in ter face Sediment/w ater in ter face Sedi ment O2 pres ent (e xces s) O2 lim iti ng D is so lv ed s ul fide present (e xc ess)[H
2S]
O xy genic photot rophi c ba ct eria (c ya nobac teria ) Aerobi c che mohe terotrophic bact eri a Chem ol it hoaut otrophic sulf ur ba ct eri a Anox yg en ic phot ot rophi c (purpl e) sul fur ba cte ri a A nae robic su lf at e-re du cing bacteria SO 4 2-H2 S S 0, S O4 2-O rg anic sul fur input het erot rophi c ba cte ri a[O
2]
G as c oncent ra ti on (% of s at urat ion) 100[O
2]
G as c oncent ra ti on (% of s at urat ion) 100 H2 SS 0, S O4 2-Figure 3 . T he dia gram of the biol og ical su lfu r cycling i n a salin e alk ali ne l ake. T he alka liphil ic, chemo lit hoa utotrop hic SOB bacteri a inh ab it the O 2 /HS - interface where two situati
ons a re possi ble: a c oexiste nce of O2 with HS - an d the presence of a
shuttle system between O
2 , F e 2+ , Mn 2+ and HS
-4. Biological oxidation of inorganic sulfur
The most abundant form of sulfur available in nature for use by living organisms is in the oxidized state (SO42-). Sulfate is biologically reduced under anaerobic conditions by sulfate-reducing bacteria (SRB) using different substrates as electron donors (organic compounds or H2). Bacterial sulfate reduction in the presence of low concentration of oxygen has also been observed (Canfield and Des Marais, 1991). The process of sulfate reduction is synonym to sulfide (H2S) production or sulfidogenesis. In a next step of biological sulfur cycle, H2S, the most reduced sulfur compound, serves as electron donor and energy source for chemolithotrophic microorganisms. The anaerobic phototrophic sulfur-oxidizing bacteria (SOB) such as Allochromatium, Chlorobium, some
Rhodospirillaceae, Ectothiorhodospiraceae and some cyanobacteria when grown
anaerobically use H2S as the electron donor for CO2-fixation. In the chemolithoautotrophic nutrition, the reduced sulfur compound has a dual role, i.e. as electron donor as is the case in the phototrophs, and as energy source. The oxidation of sulfur compounds leads to the build-up of a proton motive force, which may generate ATP for CO2-fixation. The proton motive force is also used to drive reversed electron transport to provide the reducing power (as NADH) for CO2-fixation. The CO2 fixation pathway in most known phototrophic and chemotrophic bacteria is the Calvin cycle, but also the reversed tricarboxylic acid cycle has been detected in a variety of (non)phototrophic bacteria.
Two groups of lithotrophic SOB have been distinguished previously; members of one group are able toutilize polythionates, and members of the other group are notable to do this (Kelly et al., 1997). On the basis of physiological andbiochemical data, at least two major pathways have been proposedfor different SOB: (i) the sulfur oxidationpathway and (ii) the S4 intermediate pathway involving polythionates (Kelly et al., 1997; Friedrich et al., 2001).
The product of biological sulfide oxidation is SO42- (complete oxidation), S0 (elemental sulfur) or both SO42- and S0 (incomplete oxidation). Occasionally also thiosulfate was detected as end product both under aerobic and anaerobic conditions (Jørgensen, 1990; de Zwart et al., 1996). Elemental sulfur can be excreted in the environment or it can be stored extra- or intracellularly. The forms of stored elemental sulfur were investigated recently and it was shown that sulfur atoms are associated in chains or rings to which organic radical groups are attached. In this way, the biologically stored elemental sulfur differs in structure and composition from that of sulfur deposits resulting from chemical reactions (Prange et al., 1999, 2002). The excreted and the stored elemental sulfur are further oxidized to SO42- by the same organism or by other SOB to
yield supplementary energy. Several phototrophic bacteria, when grown in the dark, can also use elemental sulfur as acceptor for electrons derived from the storage compounds. Another category of SOB is able to use H2S under anaerobic condition with NO3- as electron acceptor (denitrifying colorless sulfur bacteria). Interestingly, a number of strictly anaerobic bacteria are capable of “fermenting” partially reduced sulfur compounds, such as sulfur, thiosulfate and sulfite into a mixture of H2S and sulfate. In this way a complete turnover of inorganic sulfur compounds is possible through biological processes. When seasonal or accidental changes occur in the physico-chemical or geological parameters of the natural environments, perturbation of the sulfur cycle can follow. A predominance of sulfide production may lead to accumulation of this toxic compound, which will diffuse toward the aerobic zones. A gradient of sulfide is thus established. In a very narrow layer, at the aerobic-anaerobic interface, H2S meets O2. H2S can be oxidized either chemically or biologically under aerobic conditions. The factors that influence the rate of chemical oxidation are the concentration of the reaction components, the pH and the presence of metal ions (Kuenen, 1975). Several groups of microorganisms are able to oxidize reduced sulfur compounds such as H2S under aerobic or microaerobic conditions. Three main groups of SOB can be distinguished: the anoxygenic phototrophs (e.g. green and purple sulfur bacteria), the obligate and the facultatively autotrophic colorless sulfur bacteria among which one can find the morphologically conspicuous bacteria (Table 5). There also exist sulfur-dependent Archaea (e.g. Thermococcus, Sulfolobus, Acidianus). A group called purple nonsulfur bacteria was originally thought to be unable to use sulfide as an electron donor for the reduction of CO2 to cell material. However, under certain conditions, sulfide at low concentrations can be used by most purple nonsulfur species (Hansen and van Gemerden, 1972). Representatives of the SOB can be isolated from acidic, neutral or alkaline environments, from cold, moderate or hot habitats, as well as from low to highly saline waters and soils.
Table 5. Categories of SOB
Category Metabolic type Location
S compound used as electron donor Representatives Green sulfur bacteria Anaerobic photolithoautotrophs
Mud and anoxic water
H2S, S0 Chlorobium Purple sulfur bacteria Anaerobic or microaerophilic (photo)lithoautotrophs Oxic and anox ic water, abov e
green sulfur bact
eria la yer H2S, S2O3 2-S0 Chromatium , Rhodospirillum, Rhodobacter, Thiospirillum, Thiocapsa, Ectothiorhodospira, Halorhodospira Obligate autotrophic colorless sulfur bacteria Aerobic and anaerobic obligate chemolithoautotrophs H2S, metal sulfides, S2O32-, S0, S3O62- S4O6 2-Thiobacillus thioparus, Thermithiobacillus tepidarius, Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans, Halothiobacillus neapolitanus, Halothiobacillus halophilus, Thiomicrospira pelophila Facultatively autotrophic colorless sulfur bacteria Aerobic and anaerobic facultative chemoautorophs Soil, sed imen ts, oxic/ anoxi c inte rfaces of wat er, sulfur springs an d other volcan ic sour ces H2S, metal sulfides, S2O32-, S0, S4O6 2-Starkeya novella, Thiobacillus aquaesulis, Thiomicrospira. thyasirae, Paracoccus denitrificans, Paracoccus versutus; Morphologically conspicuous bacteria as Beggiatoa, Thiothrix, Thioploca, Achromatium, Macromonas, Thiobacterium, Thiospira, Thiomargaritha
Examples of the energy-yielding reactions used by colorless sulfur bacteria are presented in Table 6. From this table it can be noticed that, in general, the oxidation of inorganic sulfur compounds releases high amounts of energy, which is trapped as proton-motive force or sometimes, directly as ATP (by substrate-level phosphorylation). The complete oxidation of inorganic sulfur leads to production of sulfuric acid and therefore there is a strong tendency of acidification of the surrounding environment. The microbial oxidation of sulfides is important for the formation of sulfuric acid in coal mines and in sulfur deposits. The acidification resulting from the biological activity has a strong impact on large natural areas (Gonzalez-Toril et al., 2003; Lopez-Achilla et al., 2001). The sulfur-oxidizing (leading to H2SO4) and sulfate-reducing (H2S) activities of microorganisms are often related to strong corrosion in sewer systems, concrete structures and in the equipment used to mine, store or transport coal (Little et al., 2000).
Table 6. Examples of the reactions used by colorless sulfur bacteria to gain energy for growth (Robertson and Kuenen, 1992)
H2S+2O2 Î H2SO4 2H2S+O2 Î S0 + 2H2O 2S0 + 3 O
2 + 2H2O Î 2H2SO4 Na2S2O3 + 2O2 + H2O Î Na2SO4 + H2SO4 4Na2S2O3 + O2 + 2H2O Î 2Na2S4O6 + 4NaOH 2Na2S4O6 + 7O2 + 6H2O Î 2Na2SO4 + 6H2SO4
2KSCN + 4O2 + 4H2O Î (NH4)2SO4 +K2SO4 +2CO2 5H2S + 8KNO3 Î 4K2SO4 + H2SO4 + 4N2 + 4H2O 5S0 + 6KNO
3+ 2H2O Î 3K2SO4 + 2H2SO4 + 3N2
5. Taxonomy and morphology of the obligately
chemolithoautotrophic, alkaliphilic SOB from soda
lakes
The biology of inorganic sulfur oxidation was well documented in neutral (Kuenen and Beudeker, 1982; Kuenen et al., 1992) and acidic conditions (Harrison, 1984). In the neutral hypersaline environments purple sulfur bacteria use light as energy source and inorganic sulfur compounds (H2S, S0, S2O32-) as electron donors. The inorganic sulfur oxidation by
obligate haloalkaliphilic chemolithoautotrophs was only recently discovered and investigated. The autotrophic SOB bacteria capable of oxidation of inorganic sulfur compounds at moderate to high salt concentration and at high pH can be divided into three genera: Thioalkalimicrobium (low-salt tolerant alkaliphiles), Thioalkalivibrio (extremely salt tolerant and extremely halophilic alkaliphiles) (Sorokin et al., 2001a) and
Thiomicrospira (Sorokin et al., 2002b). These genera belong to the γ subdivision of the Proteobacteria (Fig. 4). The haloalkaliphilic SOB play a crucial role in the natural sulfur-cycle in the saline, alkaline environments. A large number of alkaliphilic SOB strains have been isolated and characterized in our laboratory (Sorokin et al. 1996, 2000, 2002 a, b, 2003).
5.1. Genus Thioalkalimicrobium
The genus Thioalkalimicrobium comprises species with a low DNA G-C content (48-51 mol%) isolated from the low saline Siberian soda lakes, from Kenyan soda lakes and from the saline and alkaline Mono Lake in U.S.A. The cells are rod-shaped, vibroid, spirilloid and coccoid. Some strains include motile cells with one to three polar flagella while other strains are non-motile. The cells of Thioalkalimicrobium sp. survive 4-12 months at 4oC. They can be cultivated on alkaline thiosulfate agar medium and the colonies are reddish, without sulfur deposition. The ultrastructural study of Thioalkalimicrobium cells showed a similar organization in all strains, with an undulating cell wall of the Gram-negative type and multiple carboxysome-like structures localized in the central region of the cell. The cell wall in these bacteria was very unstable under low-osmotic conditions and during storage.
The 16S rRNA gene sequence analysis of the type strains revealed that the
Thioalkalimicrobium group has a relatively close affiliation to the neutrophilic
sulfur-oxidizing bacteria of the genus Thiomicrospira (4 and 10% total sequence difference with
Thiomicrospira pelophila and Thiomicrospira crunogena type strains, respectively).
Based on phylogenetic analysis including 16S rDNA sequence analysis and DNA-DNA hybridization supplemented by phenotypic characterization, in the
Thioalkalimicrobium group three species have been described to date: Thioalkalimicrobium aerophilum, Thioalkalimicrobium sibiricum and Thioalkalimicrobium cyclicum.
The strains of Thioalkalimicrobium aerophilum were isolated from the water and surface sediments of Siberian soda lakes (e.g. the type strain AL 3T, DSM 13739T) and from Kenyan soda lake sediments.
Thioalkalimicrobium sibiricum type strain, AL 7T (DSM 13740T) was isolated from the sediments of Siberian soda lake in Buriatia (Russia) (Sorokin et al., 2001a).
0.05
Escherichia coli
Thiocapsa roseopersicina 4210 Balneatrix alpica CIP 103589
Thiorhodospira sibirica A12
Ectothiorhodospira marismortui DSM 4180 AKLD 2
Tv. nitratis ALJ 12Tv. versutus AL 2
Thiomicrospira crunogena ATCC 35932 Ectothiorhodospira mobilis DSM 237
Thioalkalispira microaerophila ALEN 1
Thioalkalimicrobium aerophilum AL 3 Tv. nitratireducens ALEN 2
Arsenite-oxidizing bacterium MLHE-1 Halorhodospira halophila DSM 244 Tv. thiocyanoxidans ARh 2 Uncultured clone ML623J-18 Tv. jannaschii ALM 2 ALMg 2 ALJ15 ALJ22 Tv.halophilus HL17 ALJ 24 Uncultured clone ML635J-54 Tv.paradoxus ARh 1 Tv.denitrificans ALJD Tv.thiocyanodenitrificans ARhD 1 Ectothiorhodospira vacuolata DSM 2111 Ectothiorhodospira shaposhnikovii DSM 243 Ectothiorhodospira marina DSM 241 Ectothiorhodospira haloalkaliphila 9902
Halorhodospira halochloris ATCC 35916 Halorhodospira abdelmalekii DSM 2110 Arhodomonas aquaeolei HA-1
Nitrococcus mobilis ATCC 25380 Alkalispirillum mobilis DSM 12769
ALPs2
Alcalilimnicola halodurans 34Alc AHN 1
symbiont of Solemya reidi symbiont of Solemya reidi gill
Thiothrix ramosa
Leucothrix mucor DSM 2157 Thiomicrospira kuenenii JB-A1 Hydrogenovibrio marinus JCM 7688
Thiomicrospira frisia JB-A2 Thiomicrospira chilensis Ch-1 Thiomicrospira thyasirae DSM 5322
Thiomicrospira pelophila DSM 1534 Thioalkalimicrobium cyclicum ALM 1 Thioalkalimicrobium sibiricum AL 7 100 100 96 99 100 100 100 98 100 100 100 100 100 100 100 98 100 100 100 100 100 99 100 100 100 96 97 100 T h i o a l k a l i v i b r i o Figure 4 . Phyloge netic tree d emonstrati ng p ositio n of the three ne w ge ner a of halo alk al ip
hilic SOB iso
lat ed from the so da la ke s. N um be rs on the bra nc hes in di ca te s b oo ts tr ap v al ue s (o nl y th e hi gh es t v al ue s ar e in cl u de d) . U n af fil ia te d s tr ai ns amon g the ge n us Thioalkalivibrio : extreme ly
salt tolerant strains from Mon
goli a (AL Mg 2) and Keny a (A LJ 15, ALJ 22, ALJ 24); AKLD 2 – facultative ly anaer obic nit rate-red ucin
g strain from Kulu
nda. Bar, 5% s equ enc e diver genc e.
Thioalkalimicrobium cyclicum (type strain ALM 1T, DSM 14477T) was isolated on solid agar medium from the oxygen-sulfide interface water layer of Mono Lake (California, U.S.A) (Sorokin et al., 2002a). Unlike strains of Thioalkalimicrobium sibiricum that are rather microaerophilic, the strains of Thioalkalimicrobium aerophilum grow faster under fully aerobic conditions. Another phenotypic difference is the tetrathionate-oxidizing capacity, which is present in Thioalkalimicrobium aerophilum and Thioalkalimicrobium
cyclicum and very low or absent in Thioalkalimicrobium sibiricum. Moreover, Thioalkalimicrobium aerophilum strains demand a higher sodium ion concentration for
optimal growth than Thioalkalimicrobium sibiricum strains (Sorokin et al., 2001a). The growth characteristics of Thioalkalimicrobium species are detailed later in this chapter. 5.2. Genus Thioalkalivibrio
The genus Thioalkalivibrio includes obligately autotrophic sulfur-oxidizing species with a high DNA G-C content (61.0-65.6 mol%). The Thioalkalivibrio group belongs to the
γ-Proteobacteria and has no immediate relatives among the other chemolithotrophic
members of the γ-Proteobacteria. The group bears a distant relationship to the anaerobic purple sulfur bacteriaof the genus Ectothiorhodospira.
The strains were isolated mostly from the Kenyan soda lakes, which are, in general, more alkaline and saline than the Siberian steppe lakes. To date, one species of
Thioalkalivibrio was isolated from Mono Lake (U.S.A.) (Sorokin et al., 2002a). The group
is represented mainly by vibrio-shaped bacteria with one polar fagellum. Some strains show spirilla-, rod-, filamentous rod-shaped cells or curved barrel-like-cells with thick capsules. It also may include strains with non-motile cells. The Gram-negative cell wall of
Thioalkalivibrio cells is undulating and the multiple carboxysome-like structures are
present in the center of the cells with the exception of the denitrifying species. In contrast to the Thioalkalimicrobium strains, cells of the Thioalkalivibrio strains were more resistant to osmotic shock and survived much longer during storage in liquid cultures at 40C. A substantial difference in cell fine structure was observed only in the haloalkaliphilic strains. Strain ALJ 15 presents a cell wall with multiple tubular extensions filled with electron-dense material. The cells of another haloalkaliphilic strain, ALJ 22, are surrounded by a large capsule, sometimes shared by several cells that tend to aggregate.
Genetically, as well as phenotypically, the Thioalkalivibrio group is more heterogenous than the Thioalkalimicrobium group. DNA-DNA hybridization demonstrated that it includes both highly related strains with more than 90% DNA homology and only weakly related representatives with a DNA similarity of about 30% with the other strains. The group includes strains that possessed important phenotypic differences and could not
be separated from the others based on its 16S RNA or DNA-DNA homology. There are moderate halotolerant and extremely halophilic and halotolerant strains. The latter are able to produce membrane-bound yellow carotenoids but genetically they could not be separated from the colorless strains. This is a first known example of pigment formation among the aerobic ‘colorless’ sulfur bacteria. In the haloalkaliphilic strains, the amount of carotenoid produced positively correlated with the salt concentration in the growth medium. The colored strains do not contain bacteriochlorophylls.
Several strains of Thioalkalivibrio are able to grow with thiocyanate (SCN-) as the sole energy and nitrogen source. They were isolated from soda lakes in South-East Siberia, Kenya and Egypt and classified into two separate species.
Thioalkalivibrio versutus (type strain AL 2T, DSM 13738T) includes strains of vibrio- to spirilla-shaped bacteria isolated from Kenyan and Siberian soda lakes. Two strains (ALJ 15 and ALJ 22) are halophilic, thermotolerant and produce a membrane-bound yellow pigment. The type strain was isolated from the surface sediments of a Siberian soda lake (Tuva Republic) (Sorokin et al., 2001a).
Thioalkalivibrio denitrificans (type strain ALJDT, DSM 13742T) includes a non-denitrifying strain from the Kenyan soda lakes, phenotypically similar to Thioalkalivibrio
versutus. The type strain ALJDT, isolated from sediments of soda lake Bogoria (Kenya), is a facultatively anaerobic and microaerobic denitrifier. The strain ALJDT had a relatively low DNA similarity with the non-denitrifying Thioalkalivibrio strains except strain ALJ 10 (55-58% similarity). ALJDT also had a protein profile very similar to that of the non-denitrifying strain ALJ 10. Strain ALJ 10 does not grow anaerobically with different nitrogen oxides as electron acceptors. The reason for such an obvious discrepancy between the genetic similarity and physiological difference is not clear. It might be speculated that this could be the result of the complete deletion of the DNA region responsible for denitrification in strain ALJ 10 (Sorokin et al., 2001a).
Thioalkalivibrio nitratis (type strain ALJ 12T, DSM 13741T) includes strains from Kenyan and Siberian soda lakes with a high DNA homology (80%). Unlike other species of the genus, they reduce nitrate to nitrite during growth with thiosulfate under oxygen-limiting conditions. Strain ALJ 21 produces membrane-bound yellow pigment and contains high level of cytochromes. The type strain, ALJ 12T was isolated from sediments of soda lake Nakuru (Kenya). They store sulfur intracellularly (Sorokin et al., 2001a).
Thioalkalivibrio nitratireducens (type strain ALEN 2T, DSM 14787T) was isolated from the hypersaline Lake Fazda (Wadi Natrun, Egypt) and presented barrel-shaped, coccoid, non-motile cells. Strain ALEN 2T is facultatively anaerobic, obligately alkaliphilic and moderately halophilic. The strain oxidizes thiosulfate, sulfide, polysulfide and, much
less actively, elemental sulfur and tetrathionate to sulfate. They grow anerobically in the presence of nitrate as electron acceptor and thiosulfate, sulfide or polysulfide as electron donor. The sole product of nitrate reduction is nitrite. Thioalkalivibrio nitratireducens is genetically most closely related to the thiocyanate-oxidizing species, Thioalkalivibrio
paradoxus. Its DNA G+C content is 64.8±0.5 mol% (Tmmethod) (Sorokin et al., 2003). Thioalkalivibrio jannaschii (type strain ALM 2T, DSM 14478T) was isolated from the O2-HS- interface layer of Mono Lake (California, USA), tolerates up to 4 M Na+ and produces a membrane-bound yellow pigment (Sorokin et al., 2002a).
Thioalkalivibrio thiocyanoxidans (type strain ARh 2T, DSM 13532T) differs from the other Thioalkalivibrio species by the ability to grow with thiocyanate as the sole energy, nitrogen and sulfur source, producing cyanate as an intermediate. Cells are short vibrios and each has a single polar flagellum. The extremely salt-tolerant strains produce a membrane-bound yellow pigment. The species is obligately chemolithoautotrophic. The type strain ARh 2T, isolated from a Kenyan soda lake, is a yellow-colored, extremely natronotolerant bacterium able to grow in soda brines (up to 4.3 M Na+) (Sorokin et al., 2002c).
Thioalkalivibrio paradoxus (type strain ARh 1T, DSM 13531T) cells are large, non-motile, barrel-like rods with capsules. It was isolated from the sediments of Kenyan (e.g. the type strain) and Egyptian soda lakes. The strains are obligately alkaliphilic and moderately halophilic (Sorokin et al., 2002c).
5.3. Genus Thioalkalispira
To date the genus Thioalkalispira is represented by a single obligately chemolithoautotrophic sulfur-oxidizing species Thioalkalispira microaerophila (type strain ALEN 1T, DSM 14786T). Isolated from a soda lake in Wadi Natrun, Egypt, the
Thioalkalispira spirillum-like bacterial cells are motile and with a single polar flagellum.
They contain a membrane-associated yellow pigment. The DNA G-C content of
Thioalkalispira microaerophila is 58.9±0.5 mol% (Tm), which is lower than the values
observed for all of the known haloalkaliphilic SOB of the genera Thioalkalimicrobium and
Thioalkalivibrio isolated so far. Phylogenetic analyses of the 16S rDNA sequences of strain
ALEN 1T and its closest relatives demonstrated that this strain formed a deep branch within the γ-Proteobacteria, with no obvious association to any described cluster of species/genera (Sorokin et al., 2002b).
6. Growth characteristics and sulfur-oxidizing potential
of alkaliphilic SOB from soda lakes
The growth physiology of the representatives of the genera Thioalkalimicrobium and
Thioalkalivibrio genera have been studied under substrate excess in batch culture or
substrate limitation in continuous culture, at alkaline pH and at low salt concentration (0.2-1.5 M Na+) (Sorokin et al., 2000, 2001a, b).
6.1. Growth characteristics of Thioalkalimicrobium sp. Optimal growth conditions
Species of Thioalkalimicrobium are obligate chemolithoautotrophic bacteria. In batch cultures, Thioalkalimcrobium strains grow optimally at pH values higher than 9. In pH-controlled thiosulfate-limited continuous culture, growth occurred within a pH range of 7.5-10.6, with an optimum around 10. At pH values lower than 8, most of the cells lysed. All strains are sodium-dependent, with a minimal sodium ion requirement of about 0.2-0.3 M. The upper limit of sodium ion concentration was 1.2-1.5 M.
Growth kinetics
As a general property for Thioalkalimicrobium species a maximum specific growth rate in chemostat culture of 0.33 h-1 was established, while in batch culture it varied from 0.08 to 0.22 h-1. The molar growth yield on thiosulfate in batch culture varied from 1.8 to 2.7 g protein (mol thiosulfate)-1 while in chemostat culture this value did not exceed 3.5 g protein (mol thiosulfate)-1 (Sorokin et al., 2001a).
Sulfur oxidation potential and cytochrome composition
The obligately chemolithoautotrophic Thioalkalimicrobium strains were able to grow only in the presence of thiosulfate or sulfide. Organic compounds (e.g. acetate, yeast extract) are assimilated without growth. Intermediate elemental sulfur formation could not be detected during batch cultivation. Under unfavorable pH conditions small amounts of sulfite were detected in some strains. During polysulfide oxidation in cell suspension or under severe oxygen limitation in thiosulfate- or sulfide-growing culture, production of elemental sulfur was observed. On the basis of their oxygen requirement, Thioalkalimicrobium strains have been divided into two categories: preferentially aerobic and preferentially microaerobic. Aerobic strains grew better under conditions of non-limiting oxygen supply. This category
included mostly rod-shaped bacteria isolated from the water or surface sediments of the Siberian soda lakes. The microaerobic strains mostly originated from the anaerobic sediments. They grew faster under conditions of limited oxygen supply. Some of them, however, were only slightly inhibited by forced aeration. The latter strains were represented by vibroid cells and formed compact colonies.
The Thioalkalimicrobium cells have, in general, high sulfide and
thiosulfate-dependent respiration activity, and low cytochrome c oxidase activity [0.2-0.3 mmol tetramethyl-ethyl-phenylene-diamine (TMPD) (mg protein)-1 min-1]. They contain high concentrations of cytochromes c and lower concentration of cytochromes b. In membranes of Thioalkalimicrobium aerophilum and Thioalkalimicrobium sibiricum the cytochrome c oxidase is of the cbb3 type - a cytochrome c oxidase with high affnity to oxygen (Sorokin et al., 2001a). This species was the only cytochrome c oxidase found in the neutrophilic sulfur bacterium Halothiobacillus neapolitanus W5 (Visser, 1997). All members of this group oxidized thiosulfate, sulfide and polysulfide with maximum rates at pH 9-10 and up to pH 11. The specific rates are comparable with the highest values observed in neutrophilic sulfur bacteria (Stefess, 1993; Visser, 1997). On the other hand, only a few of the
Thioalkalimicrobium strains were able to oxidize tetrathionate and most could not oxidize
elemental sulfur or did it with a very low specific activity. As the affinity constant for tetrathionate was at least one order of magnitude higher than that for thiosulfate and sulfide (about 80-100 mM) and because of the instability of tetrathionate under alkaline conditions at high concentrations, it is very unlikely that tetrathionate can serve as a natural substrate for the Thioalkalimicrobium group. In contrast, polysulfides can be regarded as specific substrates for alkaliphilic sulfur bacteria because they become chemically stable at alkaline pH. The stoichiometry of oxygen consumption and accumulation of colloidal sulfur during polysulfide oxidation indicated that only the terminal sulfur atoms of S62- were oxidized completely to sulfate by the Thioalkalimicrobium strains. These results demonstrated that inorganic polysulfur compounds like polysulfide are unlikely to be involved as intermediates of sulfide oxidation in these bacteria. Together with the complete inability to oxidize sulfite, the absence of sulfite dehydrogenase activity and the very low tetrathionate synthase activity in cell-free extracts, the results suggested that the Thioalkalimicrobium representatives oxidize sulfur compounds directly to sulfate by a mechanism similar to that found in several neutrophilic sulfur bacteria like Paracoccus versutus (Kelly, 1999). Purified sulfide dehydrogenase from Thioalkalimicrobium aerophilum oxidized sulfide via a one-electron mechanism implying the formation of a sulfide radical as immediate product. Further oxidation may proceed via some kind of enzyme-bound [S-S] intermediate with a step-by-step oxidation of sulfur atoms to sulfate (Sorokin et al., 1998).
6.2. Growth characteristics of Thioalkalivibrio species Optimal growth conditions
Strains of Thioalkalivibrio are obligately chemolithoautotrophs growing optimally at pH 10.0-10.2. Some strains are capable of growing down to 9-9.5 while others fail to grow below pH 10. Many strains of Thioalkalivibrio are halotolerant or extremely halotolerant, being able to grow between 0.6 and 4 M Na+. Some strains of Thioalkalivibrio versutus are obligate halophilic and thermotolerant, growing only above 1 M Na+ (optimum at 1-2 M) and up to 45-47°C (optimum at 40°C).
Growth kinetics
In general, the batch and continuous cultivation have shown that the maximum specific growth rate with thiosulfate is less than 0.2 h-1. Unlike Thioalkalimicrobium species, the maximum molar yield on thiosulfate in Thioalkalivibrio strains is higher (up to 8 g protein mol-1) (Sorokin et al., 2001a, b).
Sulfur oxidation potential and cytochrome composition
Thioalkalivibrio strains oxidize sulfide, thiosulfate, elemental sulfur, sulfite and
polythionates with relatively low activities within the pH range 7.0 to11.0-11.5 (optimum pH 9-10). The rates of oxygen consumption with various inorganic sulfur substrates are ranging between 0.2 to 0.8 µmol O2 (mg protein)-1 min-1. Sulfide and polysulfide oxidation by washed cells display a biphasic kinetics, a first phase attributed to fast oxidation to intermediate sulfur, and a second phase attributed to a slower oxidation of elemental sulfur to sulfate. Elemental sulfur is transiently produced at the beginning of the exponential phase in batch cultivation on thiosulfate at alkaline pH. Elemental sulfur when produced is stored in the periplasm. No sulfur formation was observed upon polysulfide oxidation in cell suspension. The end product of inorganic sulfur oxidation is sulfate. Tetrathionate is hydrolysed first to thiosulfate, elemental sulfur and sulfate in Thioalkalivibrio versutus strains. Overall, the results demonstrated that the bacteria of the Thioalkalivibrio group appear to employ a pathway of sulfide oxidation via polysulfur (sulfur or possibly polysulfide) compounds and sulfite, similar to many acidophilic and some neutrophilic sulfur-oxidizing bacteria (Kelly, 1999; Pronk et al., 1990).
Thioalkalivibrio thiocyanoxidans and Thioalkalivibrio paradoxus, isolated from
sediments of Kenyan or Egyptian lakes, are capable of utilizing thiosulfate as energy source and thiocyanate (SCN-) as sole source of energy, nitrogen and sulfur. Washed cells of
Thioalkalivibrio paradoxus were able to oxidize carbon disulfide, a unique property among Thioalkalivibrio species. Thiocyanate-oxidizing capacity is inducible in these strains. As a
result of thiocyanate degradation, cyanate (OCN-) is produced as intermediate compound. Cyanase activity, which liberates NH3, is absent in Thioalkalivibrio paradoxus and present in few strains of Thioalkalivibrio thiocyanoxidans. Cyanate is stable under alkaline conditions and it is used as nitrogen source by haloalkaliphilic strains. Interestingly,
Thioalkalivibrio paradoxus has an extremely low elemental sulfur oxidation activity and
therefore, the oxidation of thiosulfate, sulfide, polysulfide and thiocyanate results in the accumulation of elemental sulfur. Sulfite dehydrogenase activity in Thioalkalivibrio is AMP-independent, unlike sulfite dehydrogenase acitivity in Thioalkalimicrobium implying that the organisms may have substrate level ATP production. The cytochromes spectra showed that c- and b- types cytochromes are dominating in the membranes. The cytochrome c oxidase activity is relatively high, with moderate sensitivity to cyanide and with spectroscopic properties of cytochrome o. Thioalkalivibrio denitrificans is the only
Thioalkalivibrio species that contains cytochrome oxidase type aa3. The major ubiquinone in this genus is Q-8.
Thioalkalivibrio denitrificans grows best anaerobically in the presence of thiosulfate
as electron donor and nitrous oxide (N2O) as electron acceptor. Nitrite in small concentrations can also be used as electron acceptor but growth is slower than with N2O.
Thioalkalivibrio nitratis and Thioalkalivibrio nitratireducens are capable of reducing nitrate
to nitrite under microaerobic conditions. The cytochrome oxidase activity of nitrate-reducing strains was much lower than in other species. The activity of the cytochrome c oxidase in cell-free extracts of Thioalkalivibrio species other than Thioalkalivibrio nitratis is 5-50 times higher than in the members of Thioalkalimicrobium. This activity is also at least five times less sensitive to CN-. The latter confirmed the spectroscopic evidence on the different nature of cytochrome c oxidases in the two groups of these sulfur-oxidizing alkaliphiles. The high activity of cytochrome oxidases in Thioalkalivibrio is however in contrast with their low maximum respiratory activity as compared with
Thioalkalimicrobium. It is possible that the high oxidase activity may be an adaptation to
low oxygen concentration in order to lower the overall apparent affinity constant (Ks) for oxygen rather than increasing the overall (apparent) maximum respiratory capacity (Vmax). 6.3 Growth characteristics of Thioalkalispira species
Thioalkalispira strains use thiosulfate or sulfide as electron donors. Washed cells oxidize
thiosulfate, sulfide, polysulfide and elemental sulfur to sulfate. It grows optimally under micro-oxic conditions (1–2% O2 in the gas phase), whereas growth is inhibited under fully
oxic conditions. Among nitrous oxides only nitrate is used as electron acceptor but without growth. The representatives of the species are alkaliphilic and moderately halophilic bacteria growing between pH 8 and 10.4 (optimum around pH 10) and at a salt concentration between 0.3 and 1.5 M Na+ (optimum 0.5 M). The maximum growth rate (0.08 h-1) of the organism was achieved in a thiosulfate-limited micro-oxic continuous culture at pH 10 (Sorokin et al., 2002).
7. Potential applications of haloalkaliphilic SOB
Several environmental problems are caused by sulfur compounds like sulfate (pollution of surface water, acid mine drainage), SO2 (acid rain), H2S (odor problems, high toxicity, acid rain) and methylated sulfur compounds (odor problems, toxicity, climate change). The aim of sulfur biotechnology is to prevent loss of sulfur compounds to the atmosphere and to avoid complete oxidation of sulfur compounds to sulfate. Current research is therefore focused on the production of a sulfur compound, which can be easily separated from the waste streams, stored and re-used for other purposes. One of the successful processes is the production of elemental sulfur from H2S-containing gas streams by sulfur-oxidizing bacteria in the Thiopaq process (Paques BV, Balk, The Netherlands) (Fig. 5). In this system gasses can be treated by the absorption of H2S in a scrubber unit, subsequent biological oxidation of sulfide to elemental sulfur at neutral pH and separation of the sulfur and recycling of the percolation water to the scrubber (Janssen et al., 2001). A variety of gas streams (pressurized natural gas, synthesis gas, biogas and refinery gas) can be treated with this two-step process. Points for major innovation of this process are the enhancement of the stripping efficiency of H2S in the scrubber (by elevating the pH) and the reduction of the bleed stream of the aerobic reactor (by maintaining high salt conditions). Moreover, since high CO2 content is usual for H2S-containing industrial gases, use of alkaline carbonates in the scrubber instead of organic or inorganic alkali (NaOH) is beneficial for the effectiveness of H2S absorption.
The alkaliphilic sulfur-oxidizing bacteria that originate from soda lakes of Siberia (Russia) and Kenya can tolerate a very high pH (up to 10.6-11) and high salt concentrations (1-4 M Na+), making them attractive for biotechnological sulfide removal.
SCRUBBER BIOREACTOR Sulfide oxidation Biogas with H2S Gas without H2S Air out SULFUR SEPARATOR Bleed S0 Reactions: SCRUBBER: H2S + OH-ÎHS- +H2O BIOREACTOR: HS- + ½O2Î S0 +OH -Total: H2S + ½O2 Î S0 +H2O Air in
Figure 5. Block process diagram of the Thiopaq-bioscrubber and reaction mechanisms involved
8. Scope and outline of the thesis
The present PhD thesis was initiated and supported by Dutch Foundation for Applied Research (STW) in the framework of a project on “Production of S0 and removal of heavy metals from S-containing industrial waste streams with inorganic biotechnology”. This
project focused on the application of combinations of biological, physical and chemical processes to produce elemental sulfur from waste streams that contain sulfur compounds (e.g. from electrical power plants, mining industry, oil recovery plants and processes for the removal of heavy metals from polluted soil).
This research project aimed at the development of new S-processing unit operations utilizing microorganisms in combination with chemical and physical processes. These unit operations can be arranged in an optimal combination for a specific industrial application. In particular, the scope of this thesis was to characterize the physiology and growth kinetics of the newly isolated haloalkaliphilic chemolithotrophic SOB with respect to their potential use in the biotechnology of H2S removal at haloalkaline conditions. Living at high salt and high pH conditions implies special mechanisms that organisms have developed during the evolutionary selection. A close look to these adaptive mechanisms starting from population level down to biomolecules was another aim of this thesis.
The chemolithoautotrophic haloalkaliphilic SOB are a relatively new metabolic group isolated and characterized only in the past 5 years. Their taxonomy, metabolic diversity and the potential application in biological removal of toxic sulfur compounds were reviewed in Chapter 1 (Introduction) of this thesis.
In Chapter 2, the growth physiology of representatives of the genus
Thioalkalimicrobium and Thioalkalivibrio is compared. The competitive interaction
between these two groups of organisms and their survival strategy with direct implications in their ecology is also described. The aim was to study what are the rationales for the environmental occurrence of one or another group in the soda lakes.
The aim of Chapter 3 was to describe the sodium salt requirement for the growth and sulfur-oxidizing potential at alkaline conditions in Thioalkalivibrio versutus strains isolated from soda lakes. A clear distinction was made between the NaCl- and Na2CO3/NaHCO3 -requiring and tolerant strains.
Chapter 4 focuses on the effect of increasing salt concentration on the growth kinetics and possible implications on the cell energy metabolism in the alkaliphilic extremely salt-tolerant Thioalkalivibrio versutus strain ALJ 15. One of the goals of this research was to check whether in spite of the unusual high pH and salt concentration, this organism was capable of growing and oxidizing inorganic sulfur compound under environmental conditions similar towith those of neutrophilic sulfur-oxidizers. The second goal of the chapter was to characterize the growth of the organism on polysulfide as energy source, at high pH and high salt concentration, with respect to the potential industrial application and its ecological significance.
The growth physiology of an extremely salt-tolerant and facultative alkaliphile,
Thioalkalivibrio halophilus sp. nov., is presented in Chapter 5. This organism was chosen
as a model because it tolerated high concentrations of sodium carbonate and sodium chloride. It was also capable of growing well at pH 7.5 and 9.8. The hypothesis that two aqueous solutions with same Na+ concentration but containing different anionic species (HCO3- /CO32- and Cl-), resembling two types of saline environments, would have different osmotic pressure is verified theoretically and experimentally. This difference might have a direct consequence on the production of organic compatible solutes in the same organism. Life under extreme conditions of salt and pH requires certain physiological and biochemical adaptations.
The research concerning the salt-dependent compatible solutes production and membrane lipid composition of haloalkaliphilic sulfur-oxidizing bacteria is the subject of Chapter 6 of this thesis. In addition to the lipid composition analysis, the estimation of the membrane surface potential was performed by using a fluorescent lipophilic and pH indicator probe.
In the last chapter (Chapter 7) general conclusions, remarks and a discussion are presented, pointing the uniqueness and importance of the chemolithoautotrophic haloalkaliphilic SOB for fundamental as well as for applied research.
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